Abstract
Conditionally replicating adenoviruses (CRAds) armed with antitumor transgenes hold promise for cancer treatment. In previous studies, we showed that the 1504-siRNA targeting potential oncogene EphA3 was an efficient therapeutic transgene and that the telomerase reverse transcriptase promoter (TERTp) driving the CRAd was a more advanced generation of CRAd. Therefore, we combined Ad-TERTp-E1A-1504 by inserting 1504-siRNA into the CRAd to study its antitumor effects and mechanism of action, using Ad-TERTp-E1A-NC and nonreplicating adenovirus carrying 1504-siRNA as controls. Cell viability assays and ED50 studies of growth inhibition confirmed that Ad-TERTp-E1A-1504 has 3.5- and 1,400-fold greater ability to kill EphA3- and TERT-expressing tumor cells compared to Ad-TERTp-E1A-NC and Ad-ΔE1A-1504, respectively. Also, Ad-TERTp-E1A-1504 had little effect on cells that modestly expressed EphA3 and TERT such as 2BS. The antitumor efficacy of Ad-TERTp-E1A-1504 was also validated in vivo. Furthermore, the virus yield of Ad-TERTp-E1A-1504 in C4-2B was ~1,000 times greater than that in 2BS. No obvious differences were observed between Ad-TERTp-E1A-1504 and Ad-TERTp-E1A-NC. Both acridine orange staining and Beclin1 protein measurements indicated that autophagy with Ad-TERTp-E1A-1504 at 5 and 10 MOI was higher than that of Ad-TERTp-E1A-NC. Finally, the classical negatively regulated autophagy signaling pathway, PI3K/AKT/mTOR, was suppressed (reduced phosphorylated form) in contrast to NC, and that this was mediated by 1504-siRNA. Thus, Ad- TERTp-E1A-1504 does not harm normal cells but has dual inhibiting and killing effects on TERT- and EphA3-positive tumor cells, and this effect is mediated by the AKT/mTOR signaling pathway via induction of autophagy. These data may offer a foundation for novel antitumor therapies targeting this mechanism.